In recent years, cultivated fish caused by different bacterial pathogens and effective subunit fishery vaccines prepared by outer membrane proteins have been reported (Duan et al. 2019; He et al. 2020a; He et al. 2020b; Shao et al. 2015; Armwood et al. 2019). In previous studies, we found that European eel infected by the E. anguillarum showed high mortality rate (Guo et al. 2013), and the OmpA of E. anguillarum emulsified with Freund's adjuvant protected Japanese eels (A. japonica) from being infected by E. anguillarum (Duan et al. 2019) as well as 18 crucial lncRNAs appear to play crucial roles in anti-E. anguillarum infection in European eels (A. anguilla) immunized by the OmpA vaccine (He et al. 2021). In this study, we found that the relative protection rate (RPS) was 77.7% in European eels immunized by Freund's adjuvants emulsified OmpA, higher than 44.4% when Freund's adjuvant was used only (Zhang et al. 2017) post the infection of E. anguillarum, indicating the protective effect was originated from OmpA and Freund's adjuvant. The OmpA expressed in vitro decreased the mortality of European eels by producing higher serum titers in eels (Duan et al. 2019) and resisting the colonization of E. anguillarum in the liver (He et al. 2021) as well as protecting it from being induced a cascade of severe pathological changes (Fig. 2). However, due to Freund's adjuvants emulsified in OmpA also played essential role in resisting E. anguillarum infection, this study focused on the molecular regulation and related mechanisms of OmpA involved in the protective effect through the genome-wide strand-specific RNA sequencing of the eel liver.
Strand specific RNA sequencing (ssRNA-seq) refers to the use of Illumina high fidelity Taq enzyme to save the direction information of mRNA chains into the sequencing library when building the sequencing library. Data analysis after sequencing can determine whether the transcript is from a sense or antisense DNA strand and the lncRNA can be compared in test and control groups (Fabozzi et al. 2018). Compared to ordinary transcriptome sequencing, it can more accurately count the number of transcripts and determine the structure of genes, and can find more antisense transcripts. At present, it is widely used in the research of gene structure, gene expression regulation and lncRNA function (Lu and Le 2021; Ding et al. 2019; Zhao et al. 2022). In this study, based on the genome wide ssRNA-seq, the liver of European eels in the 3 infected groups were preliminarily explored, and the AS and lncRNA were analyzed to reveal the role of solo OmpA post the challenge of E. anguillarum.
Compared to the results of Gene and mRNA clustering, the lncRNA and TUCP clustering between 3 groups was more obvious (Fig. 3), due to the function of the TUCP was unknown, lncRNA was selected as optimum transcript to study the RNA-seq difference between the OmpA_inf and FCIA_inf group (He et al. 2021). The 37 significantly regulated lncRNAs (12 upregulated and 25 downregulated) in the comparison of OmpA_inf vs FCIA_inf (Fig. 4) were highlighted to explore the role of OmpA (Duan et al. 2019), thus to clarify the role of antigen and adjuvant in fishery vaccine respectively. In this study, RNA-seq was used for the first time to analyze the molecular mechanism of outer membrane protein antigen in fishery vaccine (Zhou et al. 2021), and the results in this study are of great significance to the development of fishery vaccines.
The GO and KEGG enrichment analysis of genes targeted by DE-lncRNAs help us to find key terms and pathways activated by the antigen of OmpA in the compare of OmpA_inf vs FCIA_inf. The results showed membrane part was the only enriched GO term in co-expression and co-location (Fig. 5A, 5B), indicating immunization of OmpA probably protect the host cell membrane from being invaded by E. anguillarum. The higher anti-OmpA antibody in the serum of eels (Duan et al. 2019; Guo et al. 2020) in OmpA_inf group would combinate with OmpA of E. anguillarum and then prevent the combination of E. anguillarum and host cells. The 56 of 293 DAS genes between OmpA_inf and FCIA_inf group involved in the GO term of membrane part (Fig. 7C) also supported it. For enriched KEGG pathways, DE-lncRNAs target 200 genes in co-expression involved in the neuroactive ligand-receptor interaction, and target 16 genes in co-location involved in herpes simplex infection in the compare of OmpA_inf vs FCIA_inf (Fig. 5C, 5D). The KEGG pathways of neuroactive ligand-receptor interaction and herpes simplex infection can actively participate in anti-infection at the early stage of bacterial and/or viral infection (Wang et al. 2021; Zhou et al. 2022), then these DE-lncRNAs elicited by OmpA immunization play an important role in the process of resisting the invasion of E. anguillarum. The 31 and 27 DAS genes between OmpA_inf and FCIA_inf group which involved in viral and bacterial infectious diseases (Fig. 7D) also directing the effect of anti-E. anguillarum infection.
In 7187 AS genes, only 293 of them were differentially (FDR < 0.05) regulated AS genes between two groups of FCIA_inf and OmpA_inf (Table 2), indicating although OmpA caused extensive AS of genes, it only has a significant impact on the AS of a small number of genes. However, compared to the Con_inf group, 620 of 15534 AS genes were DAS gens in the OmpA_inf group (data not show). Considering OmpA emulsified with Freund's adjuvant in the vaccines, the Freund's adjuvant activated 327 DAS (620 − 293) and 8347 (15534 − 7187) AS if the two components in the vaccine are additive. In fact, 596 of 12231 AS were DAS gens between two groups of FCIA_inf and Con_inf (data not show). Therefore, the two components in the vaccine are likely to have synergistic rather than additive effects, then the necessity of adding Freund's adjuvant to subunit vaccine was explained by AS genes in this study. Moreover, it is the first report on the relationship between antigen and adjuvant in vaccines from the perspective of AS analysis (Shen et al. 2022). The results of this study can provide new insight in the analysis of immune response mechanism and immune effect after vaccination. Most of the 293 DAS genes mainly involved in the GO terms of binding, cell part, cellular process, catalytic activity and membrane part which correlated closely with cell function and anti-infection (Ma et al. 2022). Furthermore, most of these genes positively get involved in the KEGG pathways of anti-bacterial or viral pathogens (Yang et al. 2021).
To confirm the RNA-seq results, 22 transcripts were selected for qRT-PCR verification. Although 20 transcripts were basically kept at the same level, there were 2 up regulated transcripts in the RNA-seq were showed the contrary results by qPCR verification (Fig. 8). Primers used in qPCR is actually designed based on the level of mRNAs, but RNA-seq can be used for differential analysis at both gene and transcripts level. For the same gene, qPCR is the difference in the expression level of a single transcript between samples, while RNA-seq is the comprehensive result of the expression level of multiple transcripts due to alternative splicing of a gene. Compare to the Con_inf group, 11 transcripts increased more than 10 times in the FCIA_inf and OmpA_group (Fig. 4A), indicating these mRNA related proteins probably involved in immunity in eels (He et al. 2020a; Wu et al. 2013; Kupsco and Schlenk 2017).
All 66 of 293 DAS genes formed a total of 50 degrees in 20 networks, but the difference of these 66 genes expression (fpkm) was not significant (log2FC = -1.1 to 1.1, padj > 0.17) between OmpA_inf and FCIA_inf group. However, the significant difference of AS in 66 genes showed by < 0.01 of the FDR values (Supplementary Table 7), indicating that gene expression is not the only factor to measure gene function, and AS may play an indispensable role (Tan et al. 2018; Tan et al. 2019; Chauhan et al. 2019). The conclusion can be deduced from the fact that although there are few genes with significant differences in expression between the two groups, the relative protection rate shows distinct differences (Fig. 1). Moreover, the proteins expressed by these DAS genes also interacted with each other. Several proteins, such as ncor2, esr1, sin3aa and ncoa2, can directly interact with four or more proteins. Since esr1 has an important function in regulating the expression of a variety of immune related proteins (Xiao et al. 2022; Kovats 2015; Millas and Duarte 2021), these proteins play an essential role in the process of host anti-pathogen infection (Khairy et al. 2018; Jha et al. 2022).
The interactions between 33 lncRNAs and 194 genes with pvalue < 0.05 were also analyzed in this study (Supplementary Fig. 1). Due to only 35 DEGs with padjust < 0.05 were found in the compare of OmpA_inf vs FCIA_inf, we used these 194 genes to construct the network (Fig. 4). Two DEG of 118213700 and 118227846 which expressed the protein of galactosyltransferase 1 and leucine zipper were targeted by 2 key DE-lncRNAs of TCONS_00383992 and TCONS_00228065, and the 2 proteins involved in the immunosuppression (Molyneux et al. 2017; Dubey et al. 2021). Downregulation of the 2 above-mentioned DE-lncRNAs decreased the expression of 2 proteins as well as relieved the immunosuppression in this study, explaining the higher RPS in the Omp_inf group than the FCIA_inf group. Moreover, the downregulated DEG of 118209822 targeted by TCONS_00285298 can expressed Galectin-4 which could improve immune response against bacterial infection by mediating pathogen recognition and opsonization (Niu et al. 2020), and a downregulated DEG of 118216371 expressing pentraxin fusion protein targeted by the TCONS_00384583 can alter immune response in green sea turtles (Chelonia mydas) and can be as an indicator of adverse health outcomes (Chaousis et al. 2023). Considering the two proteins involved in pathogen recognition, the significant decrease of bacterial load in liver of the OmpA_inf group (Zhai et al. 2021) responsible for the down-regulation of these two genes. One protein of jun dimerization protein 2 expressed by the upregulated DEG of 118225818 targeted by TCONS_00100532 was involved in the suppression of insulin gene expression (Nakane et al. 2021), indicating the increasing of blood sugar under hungry conditions originated from insulin suppression in the OmpA_inf group maintained the stability of the internal environment in eels, then helping the host clearing bacterial pathogens in this study. The TCONS_00014012 target DEG of 118222244, expressed BEN domain-containing protein 6 (BEND6), was downregulated to increase self-renewal of the host (Dai et al. 2013). The T-lymphocyte surface antigen Ly-9-like protein (ly-9, CD229) was expressed by an upregulated DEG of 118227655 which targeted by 4 DE-lncRNA of TCONS_00143367, TCONS_00143376, TCONS_00143351 and TCONS_00143357 in co-location. Due to ly-9 cells can eliminate tumor propagating cells, upregulation of the gene can inhibit the tumorigenesis of liver cells in the eels of Omp_inf group.
In conclusion, the present study confirmed that compared to the Con_inf group, the OmpA_inf group has a higher RPS than the FCIA_inf group, and the pathological changes of liver cells and blood vessels in OmpA_inf group were also slighter than those in the FCIA_inf group. Since there were only 35 DEGs between the two groups of OmpA_inf and FCIA_inf, we revealed the function of the OmpA in the OmpA_inf group from the analysis of DAS and DE-lncRNAs. Therefore, the OmpA enhanced the immune function in eels probably through the regulation of DAS and DE-lncRNA, then produce higher RPS and lower pathological damage in eels of the OmpA_inf group.